• No results found

Assessment of Cooperativity in Ternary Peptide-Cucurbit[8]uril Complexes

N/A
N/A
Protected

Academic year: 2021

Share "Assessment of Cooperativity in Ternary Peptide-Cucurbit[8]uril Complexes"

Copied!
5
0
0

Bezig met laden.... (Bekijk nu de volledige tekst)

Hele tekst

(1)

&

Peptide Complexes

Assessment of Cooperativity in Ternary Peptide-Cucurbit[8]uril

Complexes

Emanuela Cavatorta, Pascal Jonkheijm,* and Jurriaan Huskens*

[a]

Abstract: Evaluating cooperativity for cucurbit[8]uril (CB[8])-mediated ternary complexation is required for un-derstanding and advancing designs of such ternary self-as-sembled systems. A key issue is to dissect the contribu-tions of the binding steps of the first and second guest molecules to the overall ternary complex formation energy. This is addressed by performing concentration-de-pendent titrations between CB[8] and guests by means of concentration-dependent calorimetric and 1H-NMR

titra-tions. The sensitivity of the fitting of the cumulative heat of complexation of the calorimetric titrations is evaluated in terms of fitting error and enthalpy–entropy compensa-tion and, together with the NMR spectroscopic analysis of the separate species, non-cooperative binding is con-ceived to be the most probable binding scenario. The binding behavior of CB[8] homoternary complexes is simi-lar to CB[8] heteroternary complexes, with an enthalpy-driven tight fit of the guests in the CB[8] cavity overing the entropic penalty. Also for these types of com-plexes, a non-cooperative binding is the most probable.

Specific molecular recognition properties between ligands (guests) and receptors (hosts) allow non-covalent synthesis of artificial receptor–ligand complexes to occur.[1–4]

Cucurbit[n]ur-ils (CB[n]) form a new class of macrocyclic hosts that show re-markable molecular recognition properties in water.[5] The

highest affinities between CB[n]s and their guests occur when high energy solvation water molecules are released from the cavity, which generates an enthalpic gain upon complexation.[3]

CB[8] is the first homologue large enough to promote binding of two equivalents of guest forming a ternary complex.[6,7]For

example, a heteroternary complex forms through the well-de-fined sequential binding of two different guests inside the CB[8] cavity and this can drive the self-assembly of copoly-mers,[8]hydrogels,[9]particles,[10–11] and monolayers.[12] Also

ho-moternary complexes can be used for such purposes, in partic-ular as demonstrated for the binding of N-terminal aromatic amino acidic residues such as tryptophan (Trp) or phenylala-nine (Phe) to CB[8].[13] This type of CB[8]-peptide complex

ex-tends the application of CB[8] assemblies into the biological arena.[14–18]

A ternary complex offers the opportunity for tuning the as-sembly properties by cooperativity. Cooperativity describes the relationship between the affinities of binding of the first and second equivalent of guest by the host.[19] In comparison to

the affinity of the first guest molecule, the binding of the second guest can either be favored, unfavored, or unaffected (i.e., positive, negative, or non-cooperative, respectively). The principle of cooperative interactions is common in living sys-tems and modulates the function of a receptor by the concen-tration of the ligands. For example, the binding of oxygen to the four pockets of hemoglobin is a positive cooperative pro-cess resulting in an increase of the binding affinity of hemoglo-bin for the substrate oxygen upon each molecule of oxygen bound.[20] Proper design of the stability and dynamics of

self-assembled systems based on ternary interactions requires a thorough understanding of the, possibly cooperative, bind-ing behavior of the ternary complex interaction motif. In a sys-tematic study of the sequence-specific recognition of peptides by CB[8], the homoternary complex between PheGly2 and

CB[8] was proposed as a synthetic, positively cooperative re-ceptor–ligand interaction.[13] An overall ternary binding

con-stant Kter of 1.5V1011m@2 was reported for the complex

CB[8]·(PheGly2)2.[13] The positively cooperative nature of this

complex was suggested on the basis of1H-NMR experiments,

but the extent of cooperativity was not quantified.[13]Here, we

assess the degree of cooperativity for ternary complexes of CB[8] and two peptides both with an N-terminal phenylalanine, followed by either two (PheGly2) or six glycine (PheGly6)

resi-dues. Isothermal titration calorimetry (ITC) and 1H-NMR

titra-tions were used to study the dependence of the affinity of CB[8] on the concentration of the guest. A key issue is to dis-sect the contributions of the bindings of the first and second guest molecules to the overall ternary complex formation. This is addressed by performing concentration-dependent titra-tions, an evaluation of the error sensitivity in the ITC experi-ments, and by a spectroscopic analysis of the separate species by1H NMR spectroscopy.

[a] Dr. E. Cavatorta, Prof. P. Jonkheijm, Prof. J. Huskens Department of Science and Technology, University of Twente P.O. Box 217, 7500 AE, Enschede (The Netherlands) E-mail: p.jonkheijm@utwente.nl

j.huskens@utwente.nl

Supporting information, including the synthesis, concentration assessment, experimental methods for ITC and1H NMR, description of fitting model,

and summary of fitting results, for this article can be found under http:// dx.doi.org/10.1002/chem.201605284.

T 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of Creative Commons Attri-bution NonCommercial-NoDerivs License, which permits use and distribu-tion in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

(2)

Figure 1 shows the first and the second binding events be-tween the host CB[8] (H) and the peptide guest (G), leading to the formation of the 1:1 complex HG and the homoternary 1:2

complex HG2, respectively. The first equilibrium binding

con-stant K1arises from the interaction of a single guest G with the

host H. For the second binding step, the dissociation of a guest is associated with a pre-factor 2 (2*kd,2) to account for

the presence of two identical guest molecules in the cavity. Overall, the degree of cooperativity, defined by the ratio K1/K2,

governs which of the three scenarios, positive, negative, and non-cooperativity, applies, depending on whether K2 is larger

than, smaller than, or equal to1=

2K1, respectively.

An important aspect for the assessment of the degree of co-operativity is to work in an as wide as possible range of con-centrations of H and G to make use of the different concentra-tion dependencies of the binding constants for the formaconcentra-tion of HG and HG2. For a given overall binding constant Kter,

differ-ent degrees of cooperativity are expected to give differdiffer-ent spe-cies distributions. This means that the distributions of the con-centrations of H, HG, and HG2, while keeping the initial

con-centrations of host and guest constant, correspond to unique scenarios of K1/K2. To be able to accurately determine the ratio

K1/K2, different distributions of H, HG, and HG2 can be

mea-sured starting from different initial concentrations of host and guest. A proper working range of concentrations was deter-mined to be between 1 and 50 mm (see the Supporting Infor-mation for details). ITC studies were performed to determine the ratio between K1and K2for the ternary complexes of CB[8]

with the peptides PheGly2 and PheGly6. The simultaneous

fit-ting of the ITC data sets measured at three different host con-centrations provided a restricted range of physically acceptable K1/K2 ratios. Specifically, consistent with the optimal range of

concentrations, CB[8] was loaded in the cell at concentrations between 10 and 50 mm and titrated with a solution of the pep-tide guest. The enthalpograms obtained for each host–guest complex are given in Figure 2a, e. A mathematical model was used to fit the experimental heats with a least-squares minimi-zation routine (see the Supporting Information for details). Briefly, the heat of complex formation was expressed as a func-tion of the species concentrafunc-tions, and the thermodynamic pa-rameters K1, K2, DH01, and DH02 were used as fit parameters.

Heats of dilution for each set of initial concentration were also included in the model, and calculated values were confirmed by reference experiments. The best fits provided the optimal four parameters DH0

1, DH02, K1and K2, and thus the optimal K1/

K2ratio for each peptide guest. K1/K2values of around 2 were

found for both peptides (K1/K2= 2.1: 0.8 for PheGly2and 1.8:

0.4 for PheGly6, Table 1), which agrees with a non-cooperative

binding scenario.

To evaluate how sensitive the fit error is to variations of the K1/K2 ratio, the least-squares error was calculated for different

degrees of cooperativity. Thus, the parameters DH0

1, DH02, and

K2(correlated to K1) were optimized for chosen values of K1/K2.

Figure 2 shows the dependence of the fit error (Figure 2b, f) on the ratio K1/K2, and the correlated enthalpies (Figure 2c, g)

and entropies (Figure 2d, h). Figure S4 in the Supporting Infor-mation shows the changes in fit of the ITC titrations at very high and very low K1/K2. The trends in fit show, in short, that:

(a) a much higher K1/K2should be visible by a plateau of Q at

low [Gtot]/[Htot] combined with a clear inflection at [Gtot]/[Htot]= Figure 1. Equilibria of complexation of CB[8] (host, H) and peptide PheGlyn

(guest, G).

Figure 2. ITC data (markers) of binding CB[8] (H, three initial concentrations) with PheGly2(G) (a) and PheGly6(G) (e) in PBS (10 mm phosphate buffer, 2.7 mm

KCl and 137 mm NaCl, pH 7.4). ITC data (see also Figures S1–S6 in the Supporting Information) were simultaneously fitted (solid lines) to a model with K1, K2,

DH0

1, and DH02as fit parameters. Representative plots of the normalized least-squares fit error, DH0and @TDS0calculated at fixed values of the K1/K2ratio for

PheGly2(b–d) and PheGly6(f–h). Red vertical lines indicate the non-cooperative case (K1/K2=2), green areas represent the acceptable ranges of K1/K2within

(3)

1, and (b) a much lower K1/K2should lead to a rather shallow

slope at around [Gtot]/[Htot]= 1 (in Figure S4a, most visible in

the two higher concentrations, and in Figure S4b, at the two lower concentrations), which clearly conflict with the observed data. An evaluation of all thermodynamic parameters present-ed in Figure 2 allowpresent-ed for the determination of a range of pos-sible degrees of cooperativity (indicated in green in Figure 2). Values of the fit error within 20 % from the minimum error were defined as acceptable. This 20% cut-off value was select-ed basselect-ed on the variability of the minimum error observselect-ed in triplicate calorimetric experiments. Therefore, the upper boun-dary of the range of acceptable degrees of cooperativity was set at values of K1/K2 equal to 6 for PheGly2 and to 3.5 for

PheGly6. For higher values of K1/K2(strongly negative

coopera-tivity), the fit errors became quickly unacceptably high (Fig-ure 2b, f). Regarding the thermodynamic parameters, such high K1/K2ratios gave more exothermic enthalpies and less

fa-vorable entropies for the second step (Figure 2).

The lower limit of the range was determined considering that, even though the fit errors did not rise as quickly as at the upper limit, the binding enthalpies and entropies for the first and second binding events diverged more and more for values of K1/K2 lower than 0.5. Specifically, an inversion of the signs

and order of DH0

1and DH02,as well as of TDS01and TDS02,was

observed for values of K1/K2 below 0.2 for PheGly2 and below

0.1 for PheGly6 (Figure 2). Under these conditions, the second

binding event became less enthalpically favored (and more en-tropically favored) than the first step. Both steps would thus be associated with large enthalpy–entropy compensation ef-fects and opposite driving forces, that is, strongly enthalpy-driven for the first step and strongly entropy-enthalpy-driven for the second. In particular, the unfavorable positive enthalpy contri-bution (Figure 2c, g) and the highly favorably entropy (Fig-ure 2d, h) for the second step are not realistic considering that CB[8] complexation is known to be enthalpically driven and entropically unfavorable.[21–23]Overall, the considerations made

in terms of fit error and of enthalpy–entropy compensation de-termined a range of acceptable K1/K2ratios between 0.2 and 6

for PheGly2 and between 0.1 and 3.5 for PheGly6, which are

highlighted in green in Figure 2. For both peptides, these ranges indicate either a non-cooperative or a weakly, negative or positive cooperative system.

For both PheGly2and PheGly6, the second binding event has

a larger enthalpic gain than the first, as well as a larger entropy loss (Table 1). This indicates a tighter fit for the second guest in the CB[8] cavity, which is logical as it involves interaction with an already partially filled cavity. It is also in agreement with studies performed by Biedermann and co-workers[22] that

show, in the case of heteroternary complexes, a more favora-ble enthalpy for the second aromatic guest correlates with a less favorable entropy contribution. Similar to what was shown for the heteroternary complexes, this can be expected also in the case of the homoternary complexes studied here; the first guest reduces the cavity volume of CB[8] in such a way that the potential energy of the residual cavity water molecules is increased, thus leading to a stronger enthalpic re-sponse upon release of these water molecules upon the bind-ing of the second guest. In constrast, the tightly packed terna-ry complex reduces the degrees of freedom of both guests and therefore brings an additional unfavorable entropy contri-bution.[22]

Another observation from our calorimetric results is that when comparing the thermodynamic data for the two pep-tides, a stronger binding affinity was found for PheGly2with

re-spect to PheGly6, arising from differences for both the first and

second guest binding steps. In particular, the first PheGly6

seems to have a weaker interaction with the host (less favora-ble DH0

1).

Moreover, our results reveal a slightly weaker overall binding than the one reported in the literature[13]for the overall ternary

complexation of the peptide PheGly2 with CB[8] (see Kter in

Table 1), which can be explained by a higher concentration of cations competing with the guest for the binding to the host in our buffer.[25] The crystal structure of the complex[13] shows

that the shorter PheGly2can assume a circular conformation to

maximize its dipole–dipole interactions of the amidic protons with the carbonyl on the CB[8] rims. This cannot be achieved for a longer chain in the case of PheGly6, which may explain Table 1. Thermodynamic binding constants for complexes of CB[8][a]and PheGly

n. ITC PheGly2[b] ITC PheGly2[c] ITC PheGly6[b] 1H NMR PheGly2[d] 1H NMR PheGly6[d] K1/K2 2.1 (0.8) – 1.8 (0.4) 0.5 1.2 K1[M@1] 2.2 (1.1)V105 – 8.7 (0.6) V104 3.8 V105 9.2 V104 K2[M@1] 1.0 (0.2)V105 – 5.1 (1.3) V104 7.8 V105 7.7 V104 Kter[M-2][e] 2.3 (1.4)V1010 1.5 (0.2) V1011 4.4 (1.1) V109 3.0 V1011 7.1 V109 DH0 1[kcalmol@1] @11.6 (0.3) @29.6(0.2) @8.3 (0.2) – – DH0 2[kcalmol@1] @13.7 (1.7) @14.7 (2.5) – – DG0 1[kcalmol@1] @7.2 (0.3) @15.4 (0.1) @6.7 (0.1) @7.6 @6.8 DG0 2[kcalmol@1] @6.8 (0.1) @6.4 (0.2) @8.0 @6.7 TDS0 1[kcalmol@1][f] @4.3 (0.5) @14.2 (0.3) @1.5 (0.2) – – TDS0 2[kcalmol@1][f] @6.9 (2.2) @8.3 (3.3) – –

Standard deviations are given in parentheses. [a] Concentration of CB[8] was spectrophotometrically determined.[24][b] See Figure 2 and text for details.

Data obtained at 258C in PBS (10 mm phosphate buffer, 2.7 mm KCl and 137 mm NaCl, pH 7.4). [c] Data as reported[13]for the overall ternary complex HG 2.

Data based on three ITC experiments titrating 2 mm of PheGly2into 0.1 mm CB[8] in 10 mm sodium phosphate, pH 7.0 at 278C. [d] See Figure 3 and text

(4)

the observed difference in affinity. Unfortunately, the X-ray structure of the complex CB[8]·(PheGly6)2 is not available to

confirm this hypothesis. Our observations are in agreement with calorimetric experiments on heteroternary complexes of CB[8], paraquat and TrpGly2or TrpGly5that have shown a

tight-er binding for the short peptide compared to the long one.[22]

Taken together, the calorimetric data indicate that the most re-alistic scenario is the non-cooperative binding of the peptides. However, further narrowing the range of possible K1/K2

values could not be achieved by ITC alone, due to both the re-stricted operative concentration range (see above) and the convolution of the heat effects arising from the first and the second binding events. To overcome the latter limitation,1

H-NMR was used to provide direct spectroscopic insight into the (relative) concentrations of all participating species separately. This technique has a relatively low sensitivity, so fairly high concentrations are preferred; however, to prevent precipitation of CB[8], experiments were performed at 50 mm, which con-trasts an earlier study that used CB[8] at a concentration that exceeded the solubility limit.[13]A titration experiment was

per-formed at a constant total CB[8] concentration (in D2O) of

50 mm, while titrating from 0.5–4 equivalents of the peptides (Figure 3a, d and see full spectra in Figure S3 of the Support-ing Information). The three species G, HG, and HG2were

distin-guished based on the signals of the aryl protons of the guests.[13]Upon the first complexation, the upfield shifts of the

phenyl protons of the Phe residue verified the shielding of the surrounding CB[8] host molecule. With the second complexa-tion, the interaction among the two guests in the cavity of the CB[8] caused an additional upfield shift.[26] Under

non-satura-tion condinon-satura-tions for CB[8], the HG complex is well visible at low concentrations for both peptides, thus excluding a strongly positive cooperative system, in contrast to what has been de-scribed in an earlier study.[13] By monitoring the signals of the

aromatic protons (Figure 3a, d), the distributions of all species G, HG, and HG2 were determined for each titration step

(Fig-ure 3b, e). These distributions were fitted to a model express-ing the calculated distributions of species as a function of the fitting parameters K2 and K1 (see the Supporting Information

for details). The calculated data are shown as lines in Fig-ure 3b, e for the peptides PheGly2 and PheGly6, respectively.

Table 1 summarizes the values found for the optimized param-eters K1, K2, the corresponding free energies DG01, DG02 (see

also Figure S7), and the overall binding constant Kter. Higher

overall binding affinities (Kterin Table 1) were found as

expect-ed because the cations in the PBS solutions usexpect-ed for ITC can compete with the guest for the interaction with the host, thus destabilizing the complex,[25] whereas these salt effects are

absent in the solvent (D2O) used for the1H-NMR experiments.

In agreement with ITC, CB[8] binds more strongly with the shorter peptide PheGly2(3.0 V1011m@2) than the longer PheGly6

(7.1 V109m@2, Table 1). The optimal fits gave K

1/K2=0.5 and 1.2,

for PheGly2and PheGly6, respectively, indicative of

non-cooper-ative or slightly positive coopernon-cooper-ative binding.

To assess the sensitivity of the degree of cooperativity, the graphs in Figure 3c, f were obtained by optimizing K2(and the

correlated K1) at chosen values of the ratio K1/K2. The values of

the least-squares error for each K1/K2 ratio are reported for

each peptide (Figure 3c, f). A cut-off value of 20% from the minimum fit error was arbitrarily chosen to find the acceptable range of degree of cooperativity. The values of K1/K2 are in

a range between 0.2 and 1 for the shorter peptide PheGly2,

and between 0.6 and 10 for the longer PheGly6. Notably, the

minima by1H NMR are within the range of K

1/K2 obtained by

calorimetry, indicating a non-cooperative system. Taken togeth-Figure 3.1H-NMR titrations of CB[8] (50 mm) with PheGly

2(a) and PheGly6(d)

in D2O at 258C. Experimental [G] in G, HG, and HG2(data points) are

simulta-neously fitted (see also Figure S7) to a model varying K1and K2(solid lines)

for (b) PheGly2and (e) PheGly6. Plots of the normalized fit error calculated at

fixed values of the ratio K1/K2for (c) PheGly2and (f) PheGly6. Red vertical

lines in c and f indicate the non-cooperative value of K1/K2=2. Green areas

(5)

er, these results confirm a most probable scenario in which the ternary complexation between the peptides and CB[8] is non-cooperative.

It should be noted that these ternary CB[8]-peptide com-plexes cannot be compared directly to, for example, the coop-erativity observed in hemoglobin, because in the former case, the first guest does not occupy one of two identical, well-spaced binding sites, but resides somewhere in the same cavity to which also the second one binds in the next step. As a result, the second guest experiences interactions with the first guest directly, as witnessed by the correlation between en-thalpy and entropy.

In conclusion, combining the pieces of evidence from calori-metric and 1H-NMR titrations shown in this work, the most

probable scenario to describe the homoternary complexation of phenylalanine-based peptides by CB[8] is a non-cooperative mode of interaction. This is independent of the tail length of the peptides studied in this work. Remarkably, whereas the second guest experiences a stronger interaction with the host after the first complexation step, there appears to be a counter-balancing entropic contribution that leads to an overall cooperative behavior in affinity. This contrasts the normal non-cooperative behavior of well-separated binding sites, in which case the binding enthalpies of all steps are equal, and entropy differences arise solely from differences in statistical pre-fac-tors. The binding behavior of the homoternary peptide com-plexes resembles that observed for heteroternary comcom-plexes. The PheGly binding motif offers the synthetic flexibility and biocompatibility of peptides, and can have an active role in natural functional structures as well, such as in nuclear mem-brane pores.[27]The insights in the complexation between

pep-tides and CB[8] allow for a rational design of more complex self-assembled systems built on this powerful interaction motif.

Acknowledgements

B. H. M. Ruel and A. Juan Ruiz del Valle are acknowledged for technical support. Starting grant from the ERC (259183) to P.J. and E.C. is acknowledged for financial support.

Conflict of interest

The authors declare no conflict of interest.

Keywords: complexation · cooperativity · cucurbit[n]uril · self-assembly · titration

[1] D. N. Reinhoudt, M. Crego-Calama, Science 2002, 295, 2403 –2407.

[2] G. V. Oshovsky, D. N. Reinhoudt, W. Verboom, Angew. Chem. Int. Ed. 2007, 46, 2366 –2393; Angew. Chem. 2007, 119, 2418– 2445.

[3] D. A. Uhlenheuer, K. Petkau, L. Brunsveld, Chem. Soc. Rev. 2010, 39, 2817 –2826.

[4] T. Aida, E. W. Meijer, S. I. Stupp, Science 2012, 335, 813– 817.

[5] a) J. Lagona, P. Mukhopadhyay, S. Chakrabarti, L. Isaacs, Angew. Chem. Int. Ed. 2005, 44, 4844 – 4870; Angew. Chem. 2005, 117, 4922 –4949; b) J. W. Lee, S. Samal, N. Selvapalam, H.-J. Kim, K. Kim, Acc. Chem. Res. 2003, 36, 621 –630; c) E. Masson, X. Ling, R. Joseph, L. Kyeremeh-Men-saha, X. Lua, RSC Adv. 2012, 2, 1213 –1247; d) S. J. Barrow, S. Kasera, M. J. Rowland, J. Del Barrio, O. A. Scherman, Chem. Rev. 2015, 115, 12320 –12406.

[6] J. Kim, I.-S. Jung, S.-Y. Kim, E. Lee, J.-K. Kang, S. Sakamoto, K. Yamaguchi, K. Kim, J. Am. Chem. Soc. 2000, 122, 540 –541.

[7] H.-J. Kim, J. Heo, W. S. Jeon, E. Lee, J. Kim, S. Sakamoto, K. Yamaguchi, K. Kim, Angew. Chem. Int. Ed. 2001, 40, 1526 –1529; Angew. Chem. 2001, 113, 1574 –1577.

[8] U. Rauwald, O. A. Scherman, Angew. Chem. Int. Ed. 2008, 47, 3950 – 3953; Angew. Chem. 2008, 120, 4014 –4017.

[9] J. R. McKee, E. A. Appel, J. Seitsonen, E. Kontturi, O. A. Scherman, O. Ikkala, Adv. Funct. Mater. 2014, 24, 2706– 2713.

[10] J. Zhang, R. J. Coulston, S. T. Jones, J. Geng, O. A. Scherman, C. Abell, Science 2012, 335, 690– 694.

[11] C. Stoffelen, J. Voskuhl, P. Jonkheijm, J. Huskens, Angew. Chem. Int. Ed. 2014, 53, 3400 –3404; Angew. Chem. 2014, 126, 3468– 3472.

[12] Q. An, J. Brinkmann, J. Huskens, S. Krabbenborg, J. De Boer, P. Jonk-heijm, Angew. Chem. Int. Ed. 2012, 51, 12233– 12237; Angew. Chem. 2012, 124, 12399–12403.

[13] L. M. Heitmann, A. B. Taylor, P. J. Hart, A. R. Urbach, J. Am. Chem. Soc. 2006, 128, 12574–12581.

[14] S. Sonzini, S. T. J. Ryan, O. A. Scherman, Chem. Commun. 2013, 49, 8779 –8781.

[15] C. Hou, J. Li, L. Zhao, W. Zhang, Q. Luo, Z. Dong, J. Xu, J. Liu, Angew. Chem. Int. Ed. 2013, 52, 5590 –5593; Angew. Chem. 2013, 125, 5700 – 5703.

[16] S. Sankaran, M. De Ruiter, J. J. L. M. Cornelissen, P. Jonkheijm, Bioconju-gate Chem. 2015, 26, 1972 –1980.

[17] E. Cavatorta, M. L. Verheijden, W. van Roosmalen, J. Voskuhl, J. Huskens, P. Jonkheijm, Chem. Commun. 2016, 52, 7146 –7149.

[18] R. P. G. Bosmans, J. M. Briels, L. G. Milroy, T. F. A. de Greef, M. Merkx, L. Brunsveld, Angew. Chem. Int. Ed. 2016, 55, 8899– 8903; Angew. Chem. 2016, 128, 9045 – 9049.

[19] G. Ercolani, J. Am. Chem. Soc. 2003, 125, 16097 – 16103.

[20] G. K. Ackers, M. L. Doyle, D. Myers, M. A. Daugherty, Science 1992, 255, 54–63.

[21] F. Biedermann, V. D. Uzunova, O. A. Scherman, W. M. Nau, A. De Simone, J. Am. Chem. Soc. 2012, 134, 15318– 15323.

[22] F. Biedermann, M. Vendruscolo, O. A. Scherman, A. De Simone, W. M. Nau, J. Am. Chem. Soc. 2013, 135, 14879 –14888.

[23] Z. Miskolczy, L. Biczjk, Phys. Chem. Chem. Phys. 2014, 16, 20147 – 20156. [24] S. Yi, A. E. Kaifer, J. Org. Chem. 2011, 76, 10275 –10278.

[25] X. Ling, S. Saretz, L. Xiao, J. Francescon, E. Masson, Chem. Sci. 2016, 7, 3569 –3573.

[26] T. Zhang, S. Sun, F. Liu, J. Fan, Y. Pang, L. Sun, X. Peng, Phys. Chem. Chem. Phys. 2009, 11, 11134 –11139.

[27] S. Frey, R. P. Richter, D. Gçrlich, Science 2006, 314, 815–817.

Manuscript received: November 13, 2016 Accepted Article published: February 13, 2017 Final Article published: March 14, 2017

Referenties

GERELATEERDE DOCUMENTEN

Dat verbaast niet omdat de private toezichthouders in de bouwsector meestal aangesproken worden door partijen waar zij een directe werkrelatie mee hebben, terwijl de publieke

This so-called reverse recovery current [4,5] of the diode gives a direct indication how long it takes to remove the excess carriers from the central region of

In dit onderzoek zal vooral de relatie tussen Turkije en Syrië onderzocht worden om toe te werken naar de analyse van het verdrag dat deze twee landen in 1987 sloten en dat in

Based on this qualitative study on incident command decision-making, it can be concluded that cognitive load seems to affect the way duty officers make

My research question is thus framed as follows: What are the ethical dimensions of the power relations between the researcher and the research participant in a study which seeks

Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication:.. • A submitted manuscript is

(a) Birds of group I pre-exposed monocularly right- eyed for 3 h in a magnetic field of 92 µT, twice the intensity of the geomagnetic field, and then were tested in that field;

In this paper, we extend the adaptive EVD algorithm for TDE to the spatiotemporally colored noise case by using an adaptive generalized eigen- value decomposition (GEVD) algorithm or